Alpha-fetoprotein peptides

ABSTRACT

The invention relates to compounds that include peptides that inhibit estrogen receptor dependent cell proliferation. The compounds of the invention are useful for treating cell proliferative disorders or physiological conditions characterized by undesirable or unwanted estrogen induced cell proliferation, including breast cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of non-provisionalapplication Ser. No. 11/678,784 filed on Feb. 26, 2007, which claimspriority to U.S. provisional application Ser. No. 60/776,644 filed Feb.24, 2006, the disclosures of which are hereby incorporated by reference.

GOVERNMENT SUPPORT

The present application was made with support from the NationalInstitutes of Health Grant Nos. 5R01 CA102540 and R15CA 115524 andDepartment of Defense Grant Nos. W81XWH-04-1-0486 and BC031067; NationalScience Foundation Grant Nos. CHE-0457275, CHE-0116435 and CHE-0521063.The U.S. Government has certain rights in this invention.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The official copy of the sequence listing is submitted concurrently withthe specification as a text file via EFS-Web, in compliance with theAmerican Standard Code for Information Interchange (ASCII), with a filename of 410018B_Sequence Listing.txt, a creation date of Jan. 16, 2009,and a size of 11.7 KB. The sequence listing filed via EFS-Web is part ofthe specification and is hereby incorporated in its entirety byreference herein.

FIELD OF THE INVENTION

The invention relates to peptides derived from alpha-fetoprotein andtheir use to treat and/or prevent cancer, including breast cancer,glioblastoma, malignant and benign tumors in the ovaries, uterus,prostate, and thyroid.

BACKGROUND OF THE INVENTION

Alpha-fetoprotein (AFP) is an embryo specific serum alpha-globulinglycoprotein that is synthesized in the fetal yolk sac and circulatesthrough the serum of pregnant women (G.I. Abelev 1971). In the lastseveral decades, clinical researchers have investigated the potentialanti-estrogen and anti-breast cancer activities of AFP (Jacobson et al.1990). A number of studies have since shown its effectiveness as atherapeutic agent to treat estrogen-dependent breast cancer, as well asits ability to prevent pre-malignant foci from developing into breastcancer. Specifically, these studies indicate that alpha-fetoprotein(AFP) interferes with estrogen-dependent responses, including thegrowth-promoting effects of estrogen on breast cancer (Bennett et al.1998). U.S. Pat. Nos. 5,674,842 and 5,707,963 relate to a 34-amino acidpeptide derived from alpha-fetoprotein that was shown to exhibitanti-estrotrophic activity. Subsequently, an 8-amino acid stretch ofAFP, EMTPVNPG, (SEQ ID NO. 1), referred to as peptide P472-2, has wasidentified as possessing anti-estrotrophic activity (Mesfin et al.2000). Furthermore, U.S. Pat. No. 6,818,741 describes a peptide of eightto twenty amino acids, including a cyclic peptide (9-mer) that is usefulin reducing estrogen-stimulated growth of cells.

Previous efforts to identify a peptide under eight residues resulted inthe loss of anti-estrotrophic activity (DeFreest et al. 2004.) To date,the specific binding mechanism of AFP and of P472-2 is not known, makingrational development of lead compounds difficult.

SUMMARY OF THE INVENTION

The present invention provides small peptides, in some embodiments fourto seven amino acids in length that are derived from an active site ofalpha-fetoprotein. Surprisingly, the peptides of the present inventionretain the anti-estrotrophic activity of the 34-mer (P447) and 8-mer(P472-2) previously identified. It also provides for a unique,previously undisclosed modification of amino acids within the activesite of AFP, namely TOVNOGNEK.

In one aspect, therefore, the present invention relates to a syntheticpeptide four to seven amino acids in length, wherein the peptidecomprises an amino acid sequence of

-   -   a) AA₁-AA₂-AA₃-N, wherein AA₁ is threonine, serine, valine or        alanine; AA₂ is proline, hydroxyproline or serine; and AA₃ is        valine, isoleucine, leucine or threonine;    -   b) AA′₁-AA′₂-AA′₃-N-AA′₄, wherein AA′₁ is threonine, serine,        valine or alanine; AA′₂ is proline, hydroxyproline or serine;        AA′₃ is valine, isoleucine, leucine or threonine; and AA′₄ is        proline or hydroxyproline; or    -   c) AA₁-AA₂-N-AA₃, wherein AA″₁ is proline, hydroxyproline or        serine; AA″₃ is valine, isoleucine, leucine or threonine; and        AA″₃ is proline or hydroxyproline;        and wherein the peptide has anti-estrotrophic activity.

In another aspect, the invention relates to a peptide comprising anamino acid sequence selected from TPVN (SEQ ID NO.: 2), TOVN (SEQ IDNO.: 3), TPVNP (SEQ ID NO.: 4), TOVNP (SEQ ID NO.: 5), TPVNO (SEQ IDNO.: 6), TOVNO (SEQ ID NO.: 7), PVNPG (SEQ ID NO.: 8), OVNOG (SEQ IDNO.: 9), KTOVN (SEQ ID NO.: 11), KTOVNO (SEQ ID NO.: 36), KOTVNOG (SEQID NO.: 35), KTPVNPG (SEQ ID NO.: 38) and TOVNOGNEK (SEQ ID NO.:).

In yet another aspect, the invention relates to a synthetic linearpeptide comprising the amino acid sequence TOVNOGNEK (SEQ ID NO.: 34)

In a related aspect, the invention relates to analogs of the peptides ofthe invention, polymeric or multimeric forms of the peptides that retainthe activity of the unit peptide, as well as pharmaceutical compositionscomprising the anti-estrotrophic peptides of the invention.Additionally, peptides comprising one or more tandem repeats of an aminoacid sequence disclosed herein, are encompassed by the invention. Thetandem repeats may be separated by at least one spacer amino acid.

In another aspect, the invention relates to pharmaceutical compositionscomprising a peptide of the invention and a pharmaceutically acceptablecarrier. In yet another aspect, the invention relates to a method ofinhibiting the estrogen-dependent growth of cells, including breasttumor cells, using an anti-estrotrophic peptide of the invention.Accordingly, the peptide of the invention is useful in the treatment ofdiseases associated with estrogen-dependent growth, including breastcancer and uterine fibroids. Furthermore, the peptide of the inventionmay be used in conjunction with other breast cancer therapies, forexample, to potentiate the efficacy of tamoxifen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the effect of a peptide of the invention onestrogen-dependent tumor growth in an MCF7 xenograft model.

FIG. 2 is a graph showing the effect of a peptide of the invention ontumor volume in rats.

FIG. 3 is a graph showing the effect of a peptide of the invention onestrogen-stimulated T47D cell proliferation.

FIG. 4 is a graph showing the effects of a peptide of the invention ongrowth of prostate cancer cells (DC3).

FIG. 5 is a graph showing the dose-response profile of AFPep analogswith respect to inhibition of E₂-stimulated uterine growth.

FIG. 6 is a graph showing the dose-response profile of AFPep analogswith respect to inhibition of T47D human breast cancer cellproliferation

FIG. 7 is a graph showing the ability of peptide analogs of AFPep toblock the growth of human tumor xenografts in SCID mice.

DETAILED DESCRIPTION OF THE INVENTION

All publications, patents and other references cited herein areincorporated by reference in their entirety into the present disclosure.

The invention provides compounds including peptides and peptidomimeticsthat inhibit estrogen receptor dependent cell proliferation. Thecompounds of the invention are, therefore, useful for treating cellproliferative disorders or physiological conditions, including breastcancer, characterized by undesirable or unwanted estrogen induced cellproliferation.

In the description that follows, certain conventions will be followed asregards the usage of terminology.

The term “peptide”, as that term is know to those of skill in the art,refers to a molecule comprising two or more amino acids, generally fewerthan fifty, where the alpha-carboxylic group of one is bound to thealpha-amino group of the other. The universal one letter code known tothose skilled in the art is used herein for the identification of thetwenty basic amino acids. Additionally, the one letter code, “O” is usedherein to designate hydroxyproline, a hydroxylated derivative ofproline.

In one embodiment, the present invention encompasses peptides of 4-7amino acids in length and larger peptides comprising the basic (unit)4-7-mer, that are polymeric or multimeric forms thereof. In oneembodiment, for example, a peptide of the invention might comprisetandem repeats of a unit sequence with (SEQ ID NO.:29) or without (SEQID NO.:28) an intervening amino acid.

In another embodiment, the invention encompasses a peptide comprisingthe amino acid sequence, TOVNOGNEK (SEQ ID NO.: 34) and analogs thereof,including polymeric, multimeric and tandem repeat forms. The inventionfurther encompasses pharmaceutical compositions comprising TOVOGNEK andappropriate excipients and stabilizers.

The terms “mimetic”, “peptide mimetic” and “peptidomimetic” are usedinterchangeably, and generally refer to a peptide, partial peptide ornon-peptide molecule that mimics the tertiary binding structure oractivity of a selected native peptide or protein functional domain(e.g., binding motif or active site). These peptide mimetics includerecombinantly or chemically modified peptides, as well as non-peptideagents such as small molecule drug mimetics, as further described below.

The term “anti-estrotrophic activity” refers to the ability of thepeptides of the invention to inhibit or reduce the level ofestrogen-dependent proliferation in an estrogen receptor-positive (ER⁺)cell population. Such activity can be measured in a variety of ways,including the immature mouse uterine growth assay as described byBennett et al. 1998 and the human breast cancer xenograft assay asdescribed by Bennett et al. 1998 and Jacobson et al. 1990.

The term “prevent” as that term is understood by the person of ordinaryskill in the medical art (to which the present method claims aredirected) is not an absolute term. In the medical art it is understoodto refer to the prophylactic administration of a drug to substantiallydiminish the likelihood or seriousness of a condition. In the presentcontext, the term refers to the ability of the peptides of the inventionto lower incidence of palpable tumors, increase latency period and lowertumor multiplicity. The term is not intended to imply absoluteprotection from disease; rather it applies if there is a diminution inincidence and/or severity.

Peptides of the Invention

In one embodiment, the present invention relates to peptides four toseven amino acids in length comprising an amino acid sequence containedwithin amino acids 489-496 of human alpha-fetoprotein (Genbank accessionno. AAB58754), that is, EMTPVNPG (SEQ ID NO: 1), and analogs thereof.

In another embodiment, the present invention relates to peptides 9 ormore amino acids in length comprising the amino acid sequence TOVNOGNEK(SEQ ID NO. 34).

Additionally, multimeric forms of the peptides are also encompassed bythe invention. Multimeric forms include dimers, trimers, etc. of one ofthe peptides of the invention; the synthesis of dimeric peptides, forexample, is well known to those of skill in the art (see O'Leary andHughes, Journal of Biological Chemistry 278(28): 25738-25744, Aggarwalet al., Bioconjugate Chem. 17(2): 335-340, 2006, and Aggarwal et al.,Cancer Research, 66: 9171-9177, 2006.) Multimeric forms may be homo- orhetero-multimers, that is they comprise two or more identical peptidesor two or more different peptides, respectively.

In another embodiment, the invention provides for larger polymericpeptides that contain tandem repeats of one or more unit peptides withor without one or more spacer amino acids separating the tandem repeats.Examples are given in Table 1 below. Cyclization of some of these largerpeptides is also envisioned.

Because of their relatively small size, the peptides of the inventionmay be directly synthesized in solution or on a solid support inaccordance with conventional peptide synthesis techniques. Variousautomatic synthesizers are commercially available and can be used inaccordance with known protocols. The synthesis of peptides in solutionphase has become a well-established procedure for large-scale productionof synthetic peptides and as such is a suitable alternative method forpreparing the peptides of the invention. (See for example, Solid PhasePeptide Synthesis by John Morrow Stewart and Martin et al. Applicationof Almez-mediated Amidation Reactions to Solution Phase PeptideSynthesis, Tetrahedron Letters Vol. 39, pages 1517-1520 1998.)

Short peptide sequences, or libraries of overlapping peptides whichcorrespond to the selected regions described herein, can be readilysynthesized and then screened in assays designed to identify reactivepeptides. Alternatively, recombinant DNA technology may be employedwherein a nucleotide sequence which encodes a peptide of the inventionis inserted into an expression vector, transformed or transfected intoan appropriate host cell and cultivated under conditions suitable forexpression. Methods for production of a peptide by recombinant DNAtechnology are well known to those of skill in the art.

In some embodiments of the peptide of the present invention, either oneor both prolines corresponding to P475 and P478 of AFP is replaced withhydroxyproline (O). Substitution of the P475 proline with serine is alsopermissible without loss of activity. The threonine at position T474 maybe substituted with serine, valine or alanine. Valine at position V476may be substituted with isoleucine, leucine or threonine. Examples ofsome of the embodiments of the invention are given in Table 1.

TABLE 1 TPVN SEQ ID NO.: 2 TOVN SEQ ID NO.: 3 TPVNP SEQ ID NO.: 4 TOVNPSEQ ID NO.: 5 TPVNO SEQ ID NO.: 6 TOVNO SEQ ID NO.: 7 PVNPG SEQ ID NO.:8 OVNOG SEQ ID NO.: 9 PVNP SEQ ID NO.: 10 KTOVN SEQ ID NO.: 11 VNOG SEQID NO.: 12 OVNO SEQ ID NO.: 13 SPVNP SEQ ID NO.: 14 SOVNP SEQ ID NO.: 15SPVNO SEQ ID NO.: 16 SOVNO SEQ ID NO.: 17 VPVNP SEQ ID NO.: 18 VOVNP SEQID NO.: 19 VPVNO SEQ ID NO.: 20 VOVNO SEQ ID NO.: 21 APVNP SEQ ID NO.:22 AOVNP SEQ ID NO.: 23 APVNO SEQ ID NO.: 24 AOVNO SEQ ID NO.: 25 TSVNPSEQ ID NO.: 26 TSVNO SEQ ID NO.: 27 TPVNTPVN SEQ ID NO.: 28TPVNGGGGTPVNGGGGTPVN SEQ ID NO.: 29 TPVNOTPVNOTPVNO SEQ ID NO.: 30TPVNOGGGGTPVNOGGGG TPVNO SEQ ID NO.: 31 TPVNOKKKTPVNOKKKTPVNO SEQ IDNO.: 32 EKTOVNOGN SEQ ID NO.: 33 TOVNOGNEK SEQ ID NO.: 34 KTOVNOG SEQ IDNO.: 35 KTOVNO SEQ ID NO.: 36 KTPVNPG SEQ ID NO.: 38 MTOVNOG SEQ ID NO.:39

Peptidomimetics

In addition to the peptide compounds described herein, the inventionalso contemplates that other sterically similar compounds may beformulated to mimic the key portions of the peptide structure and thatsuch compounds may also be used in the same manner as the peptides ofthe invention. This may be achieved by techniques of molecular modelingand chemical design known to those of skill in the art.

In one embodiment, for example, the peptides of the invention aremodified to produce peptide mimetics by replacement of one or morenaturally occurring side chains of the 20 genetically encoded aminoacids (or D amino acids) with other side chains, for instance withgroups such as alkyl, lower alkyl, cyclic 4-, 5-, 6-, to 7-memberedalkyl, amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy,hydroxy, carboxy and the lower ester derivatives thereof, and with 4-,5-, 6-, to 7-membered heterocyclics. For example, proline analogs can bemade in which the ring size of the proline residue is changed from 5members to 4, 6, or 7 members. Cyclic groups can be saturated orunsaturated, and if unsaturated, can be aromatic or non-aromatic.Heterocyclic groups can contain one or more nitrogen, oxygen, and/orsulphur heteroatoms. Examples of such groups include the furazanyl,furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl,isoxazolyl, morpholinyl (e.g. morpholino), oxazolyl, piperazinyl (e.g.1-piperazinyl), piperidyl (e.g. T-piperidyl, piperidino), pyranyl,pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl,pyrimidinyl, pyrrolidinyl (e.g. T-pyrrolidinyl), pyrrolinyl, pyrrolyl,thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g. thiomorpholino),and triazolyl. These heterocyclic groups can be substituted orunsubstituted. Where a group is substituted, the substituent can bealkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl.Peptidomimetics may also have amino acid residues that have beenchemically modified by phosphorylation, sulfonation, biotinylation, orthe addition or removal of other moieties.

A variety of techniques are available for constructing peptide mimeticswith the same or similar desired biological activity as thecorresponding native but with more favorable activity than the peptidewith respect to solubility, stability, and/or susceptibility tohydrolysis or proteolysis (see, e.g., Morgan & Gainor, Ann. Rep. Med.Chem. 24, 243-252, 1989). Certain peptidomimetic compounds are basedupon the amino acid sequence of the peptides of the invention. Often,peptidomimetic compounds are synthetic compounds having athree-dimensional structure (i.e. a “peptide motif”) based upon thethree-dimensional structure of a selected peptide. The peptide motifprovides the peptidomimetic compound with the desired biologicalactivity, i.e., binding to IAP, wherein the binding activity of themimetic compound is not substantially reduced, and is often the same asor greater than the activity of the native peptide on which the mimeticis modeled. Peptidomimetic compounds can have additional characteristicsthat enhance their therapeutic application, such as increased cellpermeability, greater affinity and/or avidity and prolonged biologicalhalf-life.

Peptidomimetic design strategies are readily available in the art (see,e.g., Ripka & Rich, Curr. Op. Chem. Biol. 2, 441-452, 1998; Hruby etal., Curr. Op. Chem. Biol. 1, 114-119, 1997; Hruby & Balse, Curr. Med.Chem. 9, 945-970, 2000). One class of peptidomimetics comprises abackbone that is partially or completely non-peptide, but mimics thepeptide backbone atom for atom and comprises side groups that likewisemimic the functionality of the side groups of the native amino acidresidues. Several types of chemical bonds, e.g. ester, thioester,thioamide, retroamide, reduced carbonyl, dimethylene and ketomethylenebonds, are known in the art to be generally useful substitutes forpeptide bonds in the construction of protease-resistant peptidomimetics.Another class of peptidomimetics comprises a small non-peptide moleculethat binds to another peptide or protein, but which is not necessarily astructural mimetic of the native peptide. Yet another class ofpeptidomimetics has arisen from combinatorial chemistry and thegeneration of massive chemical libraries. These generally comprise noveltemplates which, though structurally unrelated to the native peptide,possess necessary functional groups positioned on a nonpeptide scaffoldto serve as “topographical” mimetics of the original peptide (Ripka &Rich, 1998, supra).

Pharmaceutical Compositions

The peptides of the invention are useful in a method of reducingestrogen-stimulated growth of cells by contacting the cells with thepeptide. Accordingly, the compounds of the invention can be administeredalone or as a pharmaceutical composition, systemically, regionally(e.g., directed towards an organ or tissue), or locally (e.g., directlyinto a tumor mass), in accordance with any protocol or route thatachieves the desired effect. The compounds and pharmaceuticalcompositions can be administered as a single or multiple dose each day(e.g., at a low dose), or intermittently (e.g., every other day, once aweek, etc. at a higher dose). The compounds and pharmaceuticalcompositions can be administered via inhalation (e.g., intra-tracheal),orally, intravenously, intraarterially, intravascularly, intrathecally,intraperitonealy, intramuscularly, subcutaneously, intracavity,transdermally (e.g., topical), transmucosally (e.g., buccal, bladder,vaginal, uterine, rectal, or nasal), by multiple administrations,sustained release (e.g., gradual perfusion over time) or a single bolus.Implantable devices, including microfabricated devices, foradministering drugs are well known and are also applicable fordelivering compounds of the invention to a subject.

As used herein the term “pharmaceutically acceptable” and“physiologically acceptable” includes solvents (aqueous or non-aqueous),solutions, emulsions, dispersion media, coatings, isotonic andabsorption promoting or delaying agents, compatible with pharmaceuticaladministration. A “pharmaceutical composition” or “pharmaceuticalformulation” therefore refers to a composition suitable forpharmaceutical use in a subject. The pharmaceutical compositions andformulations include a therapeutically effective amount of the compoundof the invention, for example, an effective amount of a peptide orpeptidomimetic, and a pharmaceutically or physiologically acceptablecarrier.

As will be known to the skilled artisan, pharmaceutical compositions canbe formulated to be compatible with a particular route ofadministration, systemic or local. Thus, pharmaceutical compositionsinclude carriers, diluents, or excipients suitable for administration byvarious routes.

Formulations for enteral (oral) administration can be contained in atablet (coated or uncoated), capsule (hard or soft), microsphere,emulsion, powder, granule, crystal, suspension, syrup or elixir.Conventional nontoxic solid carriers which include, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talcum, cellulose, glucose, sucrose, magnesiumcarbonate, can be used to prepare solid formulations. Supplementaryactive compounds (e.g., preservatives, antibacterial, antiviral andantifungal agents) can also be incorporated into the formulations. Aliquid formulation can also be used for enteral administration. Thecarrier can be selected from various oils including petroleum, animal,vegetable or synthetic, for example, peanut oil, soybean oil, mineraloil, sesame oil. Suitable pharmaceutical excipients include e.g.,starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice,flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerolmonostearate, sodium chloride, dried skim milk, glycerol, propyleneglycol, water, ethanol.

Pharmaceutical compositions for enteral, parenteral, or transmucosaldelivery include, for example, water, saline, phosphate buffered saline,Hank's solution, Ringer's solution, dextrose/saline, and glucosesolutions. The formulations can contain auxiliary substances toapproximate physiological conditions, such as buffering agents, tonicityadjusting agents, wetting agents, detergents and the like. Additives canalso include additional active ingredients such as bactericidal agents,or stabilizers. For example, the solution can contain sodium acetate,sodium lactate, sodium chloride, potassium chloride, calcium chloride,sorbitan monolaurate or triethanolamine oleate. Additional parenteralformulations and methods are described in Bai (1997) J. Neuroimmunol.80:65-75; Warren (1997) J. Neurol. Sci. 152:31-38; and Tonegawa (1997)J. Exp. Med. 186:507-515. The parenteral preparation can be enclosed inampules, disposable syringes or multiple dose vials made of glass orplastic.

Pharmaceutical compositions for intradermal or subcutaneousadministration can include a sterile diluent, such as water, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid, glutathione orsodium bisulfite; chelating agents such as ethylenediaminetetraaceticacid; buffers such as acetates, citrates or phosphates and agents forthe adjustment of tonicity such as sodium chloride or dextrose.

Pharmaceutical compositions for injection include aqueous solutions(where water soluble) or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. For intravenous administration, suitable carriers includephysiological saline, bacteriostatic water, Cremophor EL™ (BASF,Parsippany, N.J.) or phosphate buffered saline (PBS). The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquidpolyetheylene glycol, and the like), and suitable mixtures thereof.Fluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Antibacterial andantifungal agents include, for example, parabens, chlorobutanol, phenol,ascorbic acid and thimerosal. Isotonic agents, for example, sugars,polyalcohols such as manitol, sorbitol, sodium chloride may be includedin the composition. The resulting solutions can be packaged for use asis, or lyophilized, the lyophilized preparation can later be combinedwith a sterile solution prior to administration.

Pharmaceutically acceptable carriers can contain a compound thatstabilizes, increases or delays absorption or clearance. Such compoundsinclude, for example, carbohydrates, such as glucose, sucrose, ordextrans; low molecular weight proteins; compositions that reduce theclearance or hydrolysis of peptides; or excipients or other stabilizersand/or buffers. Agents that delay absorption include, for example,aluminum monostearate and gelatin. Detergents can also be used tostabilize or to increase or decrease the absorption of thepharmaceutical composition, including liposomal carriers. To protectfrom digestion the compound can be complexed with a composition torender it resistant to acidic and enzymatic hydrolysis, or the compoundcan be complexed in an appropriately resistant carrier such as aliposome. Means of protecting compounds from digestion are known in theart (see, e.g., Fix (1996) Pharm Res. 13:1760-1764; Samanen (1996) J.Pharm. Pharmacol. 48:119-135; and U.S. Pat. No. 5,391,377, describinglipid compositions for oral delivery of therapeutic agents).

For transmucosal or transdermal administration, penetrants appropriateto the barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art, and include, for example, fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be through nasal sprays orsuppositories (see, e.g., Sayani (1996) “Systemic delivery of peptidesand proteins across absorptive mucosae” Crit. Rev. Ther. Drug CarrierSyst. 13:85-184). For transdermal administration, the active compoundcan be formulated into ointments, salves, gels, or creams as generallyknown in the art. Transdermal delivery systems can also be achievedusing patches.

For inhalation delivery, the pharmaceutical formulation can beadministered in the form of an aerosol or mist. For aerosoladministration, the formulation can be supplied in finely divided formalong with a surfactant and propellant. In another embodiment, thedevice for delivering the formulation to respiratory tissue is in whichthe formulation vaporizes. Other delivery systems known in the artinclude dry powder aerosols, liquid delivery systems, inhalers, air jetnebulizers and propellant systems (see, e.g., Patton (1998)Biotechniques 16:141-143; Dura Pharmaceuticals, San Diego, Calif;Aradigm, Hayward, Calif.; Aerogen, Santa Clara, Calif.; and InhaleTherapeutic Systems, San Carlos, Calif.).

Biodegradable, biocompatable polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations are known to those skilled in the art. The materials canalso be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to cells or tissues using antibodies or viral coat proteins)can also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known in the art, for example, asdescribed in U.S. Pat. Nos. 4,235,871; 4,501,728; 4,522,811; 4,837,028;6,110,490; 6,096,716; 5,283,185; 5,279,833; Akimaru (1995) CytokinesMol. Ther. 1:197-210; Alving (1995) Immunol. Rev. 145:5-31; and Szoka(1980) Ann. Rev. Biophys. Bioeng. 9:467). Biodegradeable microspheres orcapsules or other biodegradeable polymer configurations capable ofsustained delivery of small molecules including peptides are known inthe art (see, e.g., Putney (1998) Nat. Biotechnol. 16:153-157).Compounds of the invention can be incorporated within micelles (see,e.g., Suntres (1994) J. Pharm. Pharmacol. 46:23-28; Woodle (1992) Pharm.Res. 9:260-265). Peptides can be attached to the surface of the lipidmonolayer or bilayer. For example, peptides can be attached tohydrazide-PEG-(distearoylphosphatidyl)ethanolamine-containing liposomes(see, e.g., Zalipsky (1995) Bioconjug. Chem. 6:705-708). Alternatively,any form of lipid membrane, such as a planar lipid membrane or the cellmembrane of an intact cell, e.g., a red blood cell, can be used.Liposomal and lipid-containing formulations can be delivered by anymeans, including, for example, intravenous, transdermal (see, e.g.,Vutla (1996) J. Pharm. Sci. 85:5-8), transmucosal, or oraladministration.

A pharmaceutically acceptable formulation can incorporate about 1% to99.9% of active ingredient (e.g., peptide or peptidomimetic). Thepharmaceutical compositions can be sterilized by conventional,well-known sterilization techniques, or can be sterile filtered.

Additional pharmaceutical formulations and delivery systems are known inthe art and are applicable in the methods and compositions of theinvention (see, e.g., Remington's Pharmaceutical Sciences (1990) 18thed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12th ed.,Merck Publishing Group, Whitehouse, N.J.; Pharmaceutical Principles ofSolid Dosage Forms, Technonic Publishing Co., Inc., Lancaster, Pa.,(1993); and Poznansky et al., Drug Delivery Systems, R. L. Juliano, ed.,Oxford, N.Y. (1980), pp. 253-315).

The pharmaceutical formulations can be packaged in unit dosage form forease of administration and uniformity of dosage. “Unit dosage form” asused herein refers to physically discrete unitary dosages foradministration to the subject to be treated; each unit contains apredetermined quantity of compound that produces a desired effect incombination with a pharmaceutical carrier or excipient.

The invention further provides kits including invention compounds andpharmaceutical formulations thereof, optionally packaged into suitablepackaging material. A kit typically includes a label or packaging insertincluding a description of the components or instructions for use invitro, in vivo, or ex vivo, of the components therein. A kit can containa collection of such components, e.g., two or more invention compoundsor an invention compound in combination with a nucleic acid damagingagent or an anti-proliferative agent.

EXAMPLES Peptide Synthesis

Peptides were synthesized using Fmoc solid-phase peptide synthesis on aPioneer Peptide Synthesis System (PerSeptive Biosystems, Inc.,Framingham, Mass.). Briefly, peptides were assembled onFmoc-PAL-PEG-PS-resin (Applied Biosystems, Inc.) from the C-terminus,reacting the deblocked N-terminus of the incoming amino acid to form anamide bond. Amino acids used in the synthesis had their N′-amino groupprotected by the 9-fluorenylmethyloxycarbonyl (Fmoc) group, which wasremoved by piperidine at the end of each cycle in the synthesis.Side-chain protecting groups of amino acids were Asn (Trt), Gln (Trt),Glu (OtBu), Hyp (tBu), Thr (tBu) which were deprotected bytrifluoroacetic acid (TFA) after peptide synthesis. The carboxyl groupof the amino acid was activated withO-(7-azabenzotriazol-1-yl)-1,1,2,3-tetramethyluroniumhexafluorophosphate (HATU).

After synthesis was completed, the resin was washed three times with100% propanol and the cleavage reaction was achieved by incubating theresin in 10 ml TFA/thioanisole/anisole/1,2-ethanedithiol (90:5:2:3) per0.5 g resin for 5 hours. The cleavage reaction mixture was filteredusing a sintered glass funnel to separate the solid resin from thepeptide solution. Filtrate volume was reduced to 1 ml by evaporationfacilitated with a gentle stream of air and the peptides wereprecipitated by addition of 15 ml dry-ice-chilled ethyl ether. Thepeptides were allowed to settle for 5 min at −80° C., and thesupernatant was aspirated. The peptides were then washed twice insimilar manner with 15 ml of ethyl acetate/diethylether (1.5:1, roomtemperature), the peptides were dissolved in deionized water, purifiedby reverse-phase HPLC, lyophilized and stored at −20° C.

The anti-estrogenic activity of each peptide was then determined usingthe immature mouse uterine growth assay, MCF-7 xenograft assay, andcancer prevention assay, all as previously described.

Immature Mouse Uterine Growth Assay

The anti-breast cancer activity of the linear and cyclic 8-mer and 9-merAFP-derived peptides is well-documented using the immature mouse uterinegrowth assay and the human breast cancer xenograft assay; there is astrong correlation between the results of these two assays. To evaluatepeptide-induced inhibition by the peptide of the present invention ofestrogen-stimulated proliferation of normal tissue, the immature mouseuterine growth assay was utilized. Briefly, 13-15 day-old Swiss/Websterfemale mice, 6-8 g in body weight, were distributed into treatmentgroups of 5 mice per group so that each group contained animals of thesame range of body weight. The peptide of the invention was injectedi.p., s.c., or p.o. into the mice. One hour later, estradiol (E₂) orvehicle control for E₂ was injected i.p. Twenty-two hours after thesecond injection, uteri were harvested, trimmed free of mesenteries, andimmediately weighed. The uterine weights were normalized to mouse bodyweights to compensate for differences in body weight among litters ofthe same age. Experiments employed a minimum of five mice per group andthe mean normalized uterine weight±SE for each group was calculated.Percent growth inhibition in a test group was calculated from normalizeduterine wet weights as described below.

${{Growth}\mspace{14mu} {inhibition}\mspace{14mu} (\%)} = {\frac{{{Full}\mspace{14mu} E_{2}\mspace{14mu} {stimulation}} - {E_{2}\mspace{14mu} {stimulation}\mspace{14mu} {in}\mspace{14mu} {test}\mspace{14mu} {group}}}{{{Full}\mspace{14mu} E_{2}\mspace{14mu} {stimulation}} - {{no}\mspace{14mu} E_{2}\mspace{14mu} {stimulation}}} \times 100\; \%}$

Differences between groups were evaluated, employing the nonparametricWicoxon ranks sum test. In all cases, growth inhibitions that weregreater than 25% were significant at P≦0.05. Results are shown in Table2.

TABLE 2 Dose No. of Activity Sequence ug/animal Assays % ± SD TOVNO (SEQID NO.: 7) 10 4 30 ± 7  1 6 33 ± 7  TPVNP (SEQ ID NO.: 4) 1 2 26 TOVN(SEQ ID NO.: 3) 1 2 29 TPVN (SEQ ID NO.: 2) 1 1 27 OVNO (SEQ ID NO.: 3)10 4 6 ± 5 1 2 15 PVNP (SEQ ID NO.: 10) 1 1 10 OVNOG (SEQ ID NO.: 9) 1 321 ± 6  KTOVN (SEQ ID NO.: 11) 1 5 18 ± 14 VNOG (SEQ ID NO.: 12) 1 1 11Cyc-(EKTOVNOGN) 14 34 ± 7  (SEQ ID NO.: 33)

Xenograft Assay

The peptides of the invention were evaluated for their ability toinhibit tumor growth in a human tumor xenograft assay. Briefly, MCF-7human breast cancer cells were obtained from ATCC® (Rockville, Md.) andwere grown in DMEM supplemented with 5% FCS, 1% non-essential aminoacids, 10 ng/ml insulin, 2 mM L-glutamine, 100 units/ml penicillin, and100 μg/ml streptomycin. MCF-7 cells from culture were solidified into afibrin clot. The tumor-containing clots were cut into pieces about 1.5mm in diameter and implanted under the kidney capsule of severe combinedimmunodeficient (ICR-SCID) mice (Taconic Farms, Germantown, N.Y.) asdescribed in Bennett et al. (Clin. Cancer Res. 1998; 4:2877-84).

Estrogen supplementation of mice was required for the growth of MCF-7tumors. Supplementation was accomplished by s.c. implantation of aSilastic tubing capsule containing solid E₂ (2 mm in length) inserted onthe day of tumor implantation. Tumor size was evaluated during survivallaparotomy using a dissecting microscope at the time of tumorimplantation and at days 15 and 30 after tumor implantation. Results areshown in FIG. 1 and are represented as the change in mean tumor volume(mm³) Intraperitoneal administration of TOVNO peptide at either 10μg/day or 1 μg/day was effective in reducing the increase in mean tumorvolume. Additionally, 100 μg/day p.o. significantly decreased theincrease in tumor volume.

Prevention Assay

The prevention study uses the methodology of Grubbs et al. (J. Natl.Cancer Inst. 71:625-628, 1983; Anticancer Res. 6:1395-1400, 1986) totest the ability of AFP peptides to prevent N-methyl-N-nitrosourea(MNU)-induced breast cancers in rats. Briefly, female rats were housedthree per cage in a room maintained at 72±2° F. and artificially lightedfor 12 hours daily. At 50 days of age, rats received a single injectionof MNU (50 mg/kg body weight) or vehicle in the jugular vein. NMU wasgiven to animals from the various treatment groups according to apredetermined randomization chart to ensure uniform distribution of thecarcinogen across the groups. Beginning 10 days after MNU exposure,treatment with AFP peptide by s.c. injection occurred once daily for 23days, a time chosen to mimic the gestation period of rats, or for loneror shorter times. The peptide was diluted in saline and was given in aninvestigator-blinded manner at doses between 0.03 and 0.27 mg/rat dailyin a volume of 0.2 ml. The control group of animals received daily 0.2ml s.c. injections of saline for the same time as AFP peptideadministration. Animals in the positive control group received only MNUtreatment and experienced the maximal number of tumors. The negativecontrol group of rats received no MNU and no AFP peptide. These animalsgenerated no spontaneous tumors through out the course of the study.Additional groups of animals received MNU

Beginning 30 days after MNU treatment, all rats were palpated twiceweekly for detection of mammary tumors, noting number, location, andsize. Tumor burden was determined noninvasively with calipers bymeasuring the long (D) and short (d) diameters. Assuming that tumorswere ellipsoid shaped, tumor volume was estimated as (π/6)(d)²(D). Allanimals were checked daily for signs of toxicity. Most studies wereterminated 100 days following MNU administration and at necropsy, tumorswere dissected weighed.

The results, as shown in FIG. 2, indicates that carcinogen (MNU)-exposedrats treated with the AFP peptide exhibited a reduction in tumor sizewhen when compared to untreated animals with the same carcinogenexposure.

FIG. 3 shows the results of a study to evaluate the effect of TOVNO onestrogen-stimulated T47D cell proliferation. T47D cells were obtainedfrom ATCC® (Rockville, Md.) and grown in culture according recommendedprotocols. Cells were treated with 1 nM estrogen or 100 nM TOVNO/1 nMestrogen. Control cells received 10% estrogen-free media. Cells weretreated for seven days. T47D cell proliferation was reduced as comparedto estrogen-stimulated cells that did not receive peptide.

The peptides of the invention were also evaluated for their ability toimpact on growth of PC3 (prostate cancer cells). PC3 cells were obtainedfrom ATCC® (Rockville, Md.) and maintained in accordance withrecommended culture conditions. The results, as shown in FIG. 4,indicate that the peptides of the invention have efficacy with respectto prostate cancer as well as breast cancer.

In other experiments, peptide analogs comprising the sequences TOVN andTPVN were assessed for their ability to inhibit E₂-stimulated growth ofmouse uterus after intraperitoneal and oral administration. The resultsare shown in TABLE 3.

TABLE 3 Inhibition ± S.E, % Immature mouse Peptide Sequence uterinegrowth T47D cell (SEQ ID. NO.) ↓ Intraperitoneal Oral Gavageproliferation  1. cyclo[EKTOVNOGN] 33 33 ± 7* 35 ± 4* 51 ± 4* 2.      EKTOVNOGN 33 30 ± 2* 32 ± 3* 41 ± 9* 3.         TOVNOGNEK 34 32± 2* 36 ± 6* 40 ± 7* 4.        KTOVNOG 35 35 ± 6* 41 ± 4* 5.       KTOVNO 36 32 ± 5* 43 ± 4* 6.        KTOVN 11 30 ± 4* 33 ± 9* 7.        TOVN 3 21 ± 4* 18 ± 2* 8.         TOVNO 7 38 ± 2* 30 ± 5* 48± 5* 9.          OVNO 13 7 ± 3 12 ± 2  8 ± 3 10.          OVNOG 9 25± 4* 24 ± 1* 11.           VNOG 12 5 ± 3 16 ± 6  12.        MTPVNPG 3718 ± 1  13.        KTPVNPG 38 34 ± 2* 14.        MTOVNOG 39 28 ± 2* 15.      EMTOV 40 1 ± 1 16.       EKTOV 41 9 ± 1 8 ± 2 17.       EKTPV 42 018.       EMTPV 43 5 ± 4 19.         TPVN 2 29 ± 1* 20.         TPVNP 427 ± 1* 21.          PVNP 10 6 ± 3 22.         PGVGQ 44 0 3 ± 1 Boldletters represent the residues that form the putative Type I beta-turn(8, 9). All peptides were administered at a dose of 1 μg/mouseintraperitoneally or 10 μg/mouse by oral gavage. In culture, T47D cellswere treated with each peptide at a concentration of 1 μM. *p < 0.05 ascompared to the group stimulated with E₂ alone. Wilcoxon Rank-Sum Test

The ability to inhibit E₂-stimulated growth of normal mouse uterus wasused as a screening assay for biological activity of AFPep analogs. Ingeneral, inhibitory activity ≧20% is considered to be biologicallysignificant. Each analog's inhibitory effect on E₂-stimulated growth wascompared to AFPep (a positive control) and to PGVGQ (a negativecontrol). PGVGQ (amino acids 478-482 of AFP) is a portion of the primarystructure of AFP near to the anti-cancer active site of AFP and iscomprised of amino acids (P, V, and G) that are important components ofAFPep. A scrambled analog of AFPep was not used because some scrambledpeptides retain low biological activity (data not shown). In Table 3,bold letters denote the sequences containing a putative Type 1beta-turn, which was postulated to be necessary for antiestrogenicactivity.

In Table 3, peptides 1-3 are AFPep and two linear 9-mer analogs. Analogs4-11 are hydroxyproline-containing peptides of varying lengths used toassess the minimum size required to maintain inhibitory activity.Analogs 12-14 are 7-mers that lack the N-terminal amino acid (E) ofAFPep. Analogs 15-18 are 5-mers from the N-terminus of EKTOVNOGN (analog2), but outside the putative pharmacophoric region. Analogs 17-21 arethe proline-containing peptides. Much of the Replica Exchange MolecularDynamics (REMD) work had modeled proline instead of hydroxyproline;hydroxyproline had been introduced to increase hydrophilicity.

Mesfin et al. showed that the minimum size required to maintainantiestrogenic activity was 8 amino acids and that a 7-mer peptide,MTPVNPG (analog 12), had substantially reduced antiestrogenicproperties. Kirschner et al. suggested that TOVN (analog 7) would retainantiestrogenic activity. In Table 3, analogs 4-11 were used to determinethe minimum size required to maintain antiestrogenic activity. Incontrast to results reported by Mesfin et al, linear analogs of AFPepthat were less than 8 amino acids maintained antiestrogenic activity.Even TOVN had weak antiestrogenic activity. However, OVNO lackedantiestrogenic activity. The most effective analog was 5-mer TOVNO(analog 8), which significantly inhibited E₂-stimulated uterine growth.

It was suggested that AFPep analogs forming putative Type I beta-turnswithin TPVN/TOVN sequence should retain antiestrogenic activity. Table 3shows that all analogs containing a putative Type 1 beta-turn (signifiedby bold-face font) significantly inhibited estrogen-stimulated growth ofimmature mouse uterus except for MTPVNPG (analog 12). Analogs notexhibiting a putative Type 1 beta-turn had little or no antiestrogeniceffect except for OVNOG (analog 10), which weakly inhibitedE₂-stimulated uterine growth.

Proline-containing peptides (analogs 17-21) replicated the activity ofhydroxyproline-containing peptides (analogs 4-11), which showed thatthere is not significant difference in biological activity when prolineis replaced by hydroxyproline.

Noting that if AFPep (cyclo[EKTOVNOGN]) were to be cleaved by atrypsin-like protease in vivo, the product would be TOVNOGNEK (analog3), we assessed the effectiveness of this analog as an antiestrogen.TOVNOGNEK significantly inhibited E₂-stimulated uterine growth and therewas no significant difference between its inhibitory activity and thatof AFPep. Analog 2 (EKTOVNOGN) was also active at this dose, whichconfirmed work done in previous studies.

REMD had suggested that MTPVNPG (analog 12 of Table 3) contains aputative Type I beta-turn and should significantly inhibit E₂-stimulateduterine growth. Earlier studies found that MTPVNPG was difficult tosynthesize by Fmoc solid-phase peptide chemistry and had littleantiestrogenic activity. In part this may have led to an earlierconclusion that EMTPVNPG was the minimum size AFP-derived peptide ableto retain antiestrogenic activity. We also found this analog difficultto synthesize by Fmoc chemistry. Therefore, Boc-solid-phase peptidechemistry was used to synthesize MTPVNPG and three related linear 7-merpeptides (KTOVNOG, KTPVNPG, and MTOVNOG; analogs 4, 13, and 14 of Table3). The purity and activity of each peptide were the same after bothmethods of peptide synthesis; KTOVNOG, KTPVNPG, and MTOVNOGsignificantly inhibited E₂-stimulated uterine growth, whereas MTPVNPGshowed significantly less activity.

The antiestrogenic properties of peptides outside the pharmacophoricregion (EMTOV, EKTOV, EKTPV, and EMTPV) (analogs 15-18) were alsoevaluated and found to have little or no antiestrogenic activity (Table3.)

The activities of three active peptides (EKTOVNOGN, TOVNOGNEK, andTOVNO), and one inactive peptide (OVNO) were further compared to AFPepfor their ability to inhibit E₂-stimulated growth of mouse uterus afteroral administration. As shown in Table 3, analogs active by theparenteral route were also active by the oral route of administration,and their activities were comparable to those of AFPep.

A number of AFPep analogs were assessed for in vitro anticanceractivity. Analogs containing the putative pharmacophore directlyinterfered with the growth stimulatory effects of estrogen on T47D humanbreast cancer cells in culture (Table 3), but they did not affect thebasal growth of these cells (data not shown). TOVN had very weakinhibitory activity against E₂-stimulated T47D cell growth. OVNO lackedinhibitory activity. TOVNO significantly inhibited E₂-stimulated T47Dhuman breast cancer cell growth and was comparable to AFPep in thisactivity (Table 3). TOVNOGNEK was also comparable to AFPep in itsability to inhibit the E₂-stimulated growth of T47D human breast cancercells.

As shown in FIG. 5 a, the linear 9-mer precursor (analog 2) of AFPepexhibited a biphasic dose-response curve in the E₂-stimulated uterinegrowth assay. Cyclization (to yield AFPep) attenuated but did notcompletely eliminate this loss of activity with increasing doses (FIG. 5b). The AFPep dose-response profile was compared to the profiles seenfrom two of the more active linear peptides, TOVNO and TOVNOGNEK. FIG. 5c shows a biphasic dose-response curve for linear TOVNO. TOVNO inhibitedE₂-stimulated uterine growth in a dose-dependent manner up to 1μg/mouse, but thereafter it lost inhibitory effect with increasing dose.FIG. 5 d shows that TOVNOGNEK inhibited E₂-stimulated uterine growth andmaintained its inhibitory effect at high doses.

FIG. 6 shows the dose response profiles of these four active peptidesagainst E₂-stimulated growth of human breast cancer cells in vitro.EKTOVNOGN inhibited E₂-stimulated T47D cell growth at low concentrationsbut lost activity at high concentrations, leading to a biphasic doseresponse curve (FIG. 6 a). AFPep inhibited the E₂-stimulated growth ofT47D cells in a concentration dependent manner up to 10⁻⁶M but lostactivity at higher concentrations (FIG. 6 b). The shorter 5-mer peptide,TOVNO, was an effective inhibitor of E₂-stimulated growth of T47D humanbreast cancer cells in a concentration-dependent manner up to 10⁻⁷M, butlost activity with increasing concentration thereafter (FIG. 6 c).However, TOVNOGNEK inhibited E₂-stimulated T47D cell growth in aconcentration dependent manner and maintained its activity withincreasing concentrations (FIG. 6 d) at concentrations as high as 10⁴ M.

The T47 D human breast cancer cell line is responsive in culture, but itwas a less reliable tool when grown as a xenograft tumor in immunedeficient mice, having a take-rate of <60% in these mice. However, theMCF-7 human breast cancer cell line has a tumor take-rate of 100% inimmune deficient mice and is completely dependent on E₂ for growth inthese mice (2). Consequently, MCF-7 xenografts were used as a model forER+ human breast cancer to evaluate, in vivo, the effectiveness of TOVNOand TOVNOGNEK as anticancer agents. As shown in FIG. 7, MCF-7 humanbreast cancer tumors were dependent on supplemental estrogen for growthas a xenograft in male SCID mice. AFPep, TOVNOGNEK, or TOVNO, each givenonce a day at a dose of 100 μg by oral gavage (FIG. 7 a) or 10 μg permouse i.p., (FIG. 7 b) significantly inhibited the growth of ER+ humanMCF-7 breast cancer tumors.

LITERATURE CITED

-   1. G. I. Abelev, Advances in Cancer Research 14, 295 (1971).-   2. H. I. Jacobson, J. A. Bennett, G. J. Mizejewski, Cancer Research    50, 415 (Jan. 15, 1990).-   3. J. A. Bennett, S. J. Zhu, A. Pagano-Mirarchi, T. A. Kellom, H. I.    Jacobson, Clinical Cancer Research 4, 2877 (November, 1998).-   4. F. B. Mesfin, T. T. Andersen, H. I. Jacobson, S. Zhu, J. A.    Bennett, Journal of Peptide Research 58, 246 (September 2001).-   5. L. A. DeFreest et al., Journal of Peptide Research 63, 409 (May,    2004).-   6. F. B. Mesfin, J. A. Bennett, H. I. Jacobson, S. J. Zhu, T. T.    Andersen, Biochimica Et Biophysica Acta-Molecular Basis of Disease    1501, 33 (Apr. 15, 2000).-   7. J. A. Bennett, F. B. Mesfin, T. T. Andersen, J. F. Gierthy, H. I.    Jacobson, Proc Natl Acad Science 99, 221T1 (February 2002).-   8. L. E. Eisele, F. B. Mesfin, J. A. Bennett, T. T. Andersen, H. I.    Jacobson, D. D. Vakharia, R. MacColl, G. J. Mizejewski, Journal of    Peptide Research 57, 539 (June 2001).-   9. L. E. Eisele, F. B. Mesfin, J. A. Bennett, T. T. Andersen, H. I.    Jacobson, H. Soldwedel, R. MacColl, G. J. Mizejewski, Journal of    Peptide Research 57, 29 (January 2001).-   10. S. Aggarwal, P. Singh, O. Topaloglu, J. T. Isaacs, S. R.    Denmeade, Cancer Research 66, 9171 (Sep. 15, 2006).-   11. S. Aggarwal, J. L. Harden, S. R. Denmeade, Bioconjugate Chem.,    17, 335 (Feb. 8, 2006).-   12. P. D. O'Leary, R. A. Hughes, The Journal of Biological    Chemistry, 278, 25738 (Jul. 11, 2003).

1-10. (canceled)
 11. A synthetic linear peptide comprising the amino acid sequence TOVNOGNEK (SEQ ID NO.: 34).
 12. The synthetic linear peptide of claim 11, wherein said peptide has anti-estrotrophic activity.
 13. A synthetic peptide comprising one or more tandem repeats of SEQ ID NO.:
 34. 14. The synthetic peptide of claim 13, wherein said tandem repeats are separated by at least one spacer amino acid.
 15. A method of reducing estrogen-stimulated growth of cells, the method comprising exposing said cells to the peptide of claim
 11. 16. The method of claim 15, comprising exposing the cells to tamoxifen before, during, or after exposing the cells to the peptide.
 17. A method for the treatment of breast cancer in a patient comprising administering to the patient a therapeutically effective amount of the peptide of claim
 11. 18. A pharmaceutical composition comprising the peptide of claim 11 and a pharmaceutically acceptable carrier. 19-24. (canceled) 